Increased fermentative adenosine production by gene-targeted Bacillus subtilis mutation

Li, Biao; Yan, Zhiying; Liu, Xiaona; Zhou, Jun; Wu, Xuezhong; Wei, Ping; Jia, Honghua; Yong, Xiaoyu

doi:10.1016/j.jbiotec.2019.04.007

Cited by 8 publications

(9 citation statements)

References 12 publications

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“…In this study, a rationally designed strategy based on the genome-scale metabolic network model succeeded in maximizing the biosynthesis of purine intermediates and biomass. With advantages over previous studies [ 6 , 7 , 9 , 11 , 13 , 14 ], the method developed here investigates the global regulation and metabolic flux to construct a general purine chassis strain for the first time and achieves the highest yield of inosine in de novo engineered bacteria to our knowledge. Based on the metabolic pathway, purine nucleosides are not the end products, but can be converted into ribose and base by the salvage pathway, resulting in a decline of nucleoside accumulation [ 4 ].…”

Section: Discussionmentioning

confidence: 99%

“…Under guidance of transcriptional and metabolite pool analysis, overexpression of prs , purF and purA in B. subtilis XGL has been reported to increase PRPP concentration and pur operon transcription, which achieved adenosine accumulation to 7.04 g/L [ 7 ]. In 2019, Li et al inactivated the GMP synthetase gene guaA and optimized the growth condition to increase adenosine titer from 7.40 to 14.39 g/L in B. subtilis A509 [ 6 ]. Besides, Escherichia coli is another industrial strain that is applied to nucleoside synthesis and has been reported to produce 6.7–7.5 g/L inosine by overexpressing the prs and purF genes with inactivation of the purA , deoD , purF , purR , add , edd , pgi and xapA genes [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

“…Currently, tumors and influenza viruses seriously threaten the health of humans, triggering increasing demands for nucleoside products worldwide. Microbial fermentation is one of the major approaches for producing purine nucleosides, and investigations have been carried out to construct genetically engineered bacteria by metabolic engineering [4,[6][7][8][9]. It has been revealed that purine nucleosides are de novo synthesized via the glycolysis pathway (EMP, Embden-Meyerhof-Parnas pathway), pentose phosphate pathway (PPP), and purine synthesis pathway (Additional file 1: Figure S1).…”

Section: Introductionmentioning

confidence: 99%

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In silico-guided metabolic engineering of Bacillus subtilis for efficient biosynthesis of purine nucleosides by blocking the key backflow nodes

Deng

Qiu

Sun

et al. 2022

Biotechnol Biofuels

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Background Purine nucleosides play essential roles in cellular physiological processes and have a wide range of applications in the fields of antitumor/antiviral drugs and food. However, microbial overproduction of purine nucleosides by de novo metabolic engineering remains a great challenge due to their strict and complex regulatory machinery involved in biosynthetic pathways. Results In this study, we designed an in silico-guided strategy for overproducing purine nucleosides based on a genome-scale metabolic network model in Bacillus subtilis. The metabolic flux was analyzed to predict two key backflow nodes, Drm (purine nucleotides toward PPP) and YwjH (PPP–EMP), to resolve the competitive relationship between biomass and purine nucleotide synthesis. In terms of the purine synthesis pathway, the first backflow node Drm was inactivated to block the degradation of purine nucleotides, which greatly increased the inosine production to 13.98–14.47 g/L without affecting cell growth. Furthermore, releasing feedback inhibition of the purine operon by promoter replacement enhanced the accumulation of purine nucleotides. In terms of the central carbon metabolic pathways, the deletion of the second backflow node YwjH and overexpression of Zwf were combined to increase inosine production to 22.01 ± 1.18 g/L by enhancing the metabolic flow of PPP. By switching on the flux node of the glucose-6-phosphate to PPP or EMP, the final inosine engineered strain produced up to 25.81 ± 1.23 g/L inosine by a pgi-based metabolic switch with a yield of 0.126 mol/mol glucose, a productivity of 0.358 g/L/h and a synthesis rate of 0.088 mmol/gDW/h, representing the highest yield in de novo engineered inosine bacteria. Under the guidance of this in silico-designed strategy, a general chassis bacterium was generated, for the first time, to efficiently synthesize inosine, adenosine, guanosine, IMP and GMP, which provides sufficient precursors for the synthesis of various purine intermediates. Conclusions Our study reveals that in silico-guided metabolic engineering successfully optimized the purine synthesis pathway by exploring efficient targets, which could be applied as a superior strategy for efficient biosynthesis of biotechnological products.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In silico-guided metabolic engineering of Bacillus subtilis for efficient biosynthesis of purine nucleosides by blocking the key backflow nodes

Deng

Qiu

Sun

et al. 2022

Biotechnol Biofuels

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“…22 Deletion of the guaA gene encoding the GMP synthase and overexpression of the purA gene in B. subtilis resulted in the production of adenosine at a titer of 14.39 g/L. 23 As PRPP is a key precursor for purine nucleoside and NMN/NR production, the high level of purine nucleoside synthesis shows that Bacillus is a promising host for NMN and NR production. But so far, Bacillus has not been developed into an excellent cell factory for producing NMN and NR.…”

Section: ■ Introductionmentioning

confidence: 99%

Metabolic Engineering of Bacillus licheniformis for the Bioproduction of Nicotinamide Riboside from Nicotinamide and Glucose

Zhou

Che

et al. 2023

ACS Sustainable Chem. Eng.

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Nicotinamide riboside (NR) is an important nucleotide, a precursor of nicotinamide adenine dinucleotide, that has attracted great attention as a promising nutraceutical to improve various symptoms of diabetes, vascular disease, and age-related physiological decline. Here, we systematically engineered Bacillus licheniformis to produce NR from glucose and nicotinamide (NAM) efficiently. Specifically, we selected the pathway using nicotinamide phosphoribosyltransferase (NAMPT) for converting NAM to nicotinamide mononucleotide (NMN) in the presence of 5-phosphoribosyl-1-pyrophosphate (PRPP) and increased the titer of NMN by engineering NAMPT and strengthening the PRPP supply. To prevent the degradation of NR, genes deoD and pupG coding purine nucleoside phosphorylase were deleted, resulting in 1.76 g/L NR accumulation in the culture medium. Then, we further deleted the nicotinamidase PncA encoding gene to prevent the synthesis of the byproduct nicotinate and overexpressed nucleotidase YfkN during the conversion of NMN to NR, where the titer of NR was increased by 226 and 41%, respectively. Notably, the efflux pump MdtL from Escherichia coli was first proven to be beneficial for NR production. Finally, combined with process optimization, the recombinant strain NR17 produced 11.33 g/L NR with a yield of 0.91 mol/mol NAM and a productivity of 0.44 g/(L•h) in a shake flask. An NR producer was constructed in this study and will be promising for the low-cost, high-quality industrial production of NR.

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“…At present, Ganoderma adenosines are mainly obtained from fruiting bodies and mycelia of G. lucidum [7]. Compared to fruiting body cultures, a submerged culture is a promising alternative for the production of adenosines, as it is quality-stable and cost-effective [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Overexpression of purine nucleoside phosphorylase increases the adenosine content in Ganoderma lucidum

Zhu

Xiao

Zhou

et al. 2022

Preprint

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Background: Adenosine has been getting increasing attention due to its positive role in immunomodulation, anti-inflammation, and anti-cancer, etc.. The low production of Ganoderma adenosine is a bottleneck for clinical trials and commercial applications. Regulating the expression of key adenosine biosynthetic gene is an optimized way to increase the production of adenosine in submerged culture of Ganoderma lucidum. Results: In this study, we correlated the expression of adenosine synthase genes (including GlATIC, GlPNP, GlADK) with the adenosine content in mycelium at different fermentation time points. The results showed that GlPNP was positively correlated with Ganoderma adenosine contents. Then the key biosynthetic gene GlPNP was cloned, characterized and overexpressed in G. lucidum. The cDNA of GlPNP gene was 969-bp in length, with a predicted molecular weight of 34.6 kDa and PI of 5.89. The GlPNP displayed a trimeric quaternary structure by theoretically modelling with SWISS-MODEL. The transcript levels of GlPNP overexpression transformants (namely OE::GlPNP-5 and OE::GlPNP-7) were approximately 2.9-3.9-fold higher than those of the WT strains on day 4, while the adenosine contents were increased by 78% and 63%, respectively, by compared with vector-containing strain. In addition, the GlPNP overexpression strains showed decreased colony growth and reduced biomass in submerged cultivation. Conclusions: GlPNP gene overexpression is an effective strategy to improve the production of adenosine in G. lucidum. This study is the first report about the manipulation of adenosine biosynthesis in medicinal fungi.

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Increased fermentative adenosine production by gene-targeted Bacillus subtilis mutation

Cited by 8 publications

References 12 publications

In silico-guided metabolic engineering of Bacillus subtilis for efficient biosynthesis of purine nucleosides by blocking the key backflow nodes

In silico-guided metabolic engineering of Bacillus subtilis for efficient biosynthesis of purine nucleosides by blocking the key backflow nodes

Metabolic Engineering of Bacillus licheniformis for the Bioproduction of Nicotinamide Riboside from Nicotinamide and Glucose

Overexpression of purine nucleoside phosphorylase increases the adenosine content in Ganoderma lucidum

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